Nonalcoholic fatty liver disease (NAFLD) is marked by accumulation of fat in liver cells with accompanying inflammation and variable degrees of cell injury and fibrosis. When cell injury and fibrosis are present, the disease has a potential to progress and is referred to as nonalcoholic steatohepatitis (NASH), which can lead to cirrhosis, liver cancer, morbidity and mortality. The etiology of NASH is not clear, nor is there an approved treatment modality for it. Our focus on NAFLD is three-fold: first, we aim to identify and refine effective treatments for the disorder. Secondly, we aim to identify and characterize key genes that play a role in the pathogenesis of NAFLD through the use of genetic studies and cell- or animal models. Our third focus is on the clinical interaction between fat accumulation in the liver and other diseases or disorders Starting in 2002, we conducted a series of clinical research studies in NASH. An initial study focused on pioglitazone, an insulin-sensitizing agent that is used widely in diabetes and in our study demonstrated significant improvement of the liver disease after one year of treatment. We also performed a prospective, open-labeled study using metformin to treat NASH. We were able to demonstrate improvement of liver disease in 30% of patients but this seemed to be secondary to metformin-induced weight loss. Vitamin E has been shown in a randomized placebo-controlled trial to be an effective therapy for NASH. Curiously, treatment with vitamin E resulted not only in a decrease in injury (thought to reflect vitamin Es antioxidant effect) but was also associated with a decrease in liver fat, through an unknown mechanism. Furthermore, the optimal dose of vitamin E to treat NASH is currently unknown. Recently, we began a new study, aimed at determining the mechanism of action of vitamin E and its optimal dose. After a period of intensive lifestyle intervention, patients undergo extensive evaluation, including imaging, metabolic phenotyping, and liver and adipose tissue sampling, and then are randomized to one of three vitamin E doses. A repeat evaluation is performed at week 4 of treatment and again, after 24 weeks. This study is currently enrolling patients. Genome wide association (GWA) studies identified single nucleotide polymorphisms (SNPs) that are associated with increased hepatic fat or elevated liver enzymes, presumably reflecting nonalcoholic fatty liver disease (NAFLD). We initiated a study to investigate whether these SNPs are associated with histological severity in a large cohort of NAFLD patients. 1117 (894 adults/223 children) individuals enrolled in NASH-Clinical Research Network and National Institutes of Health Clinical Center studies with histologically-confirmed NAFLD were genotyped for six SNPs that are associated with hepatic fat or liver enzymes in GWA studies. In adults, 3 SNPs on chromosome 22 showed associations with histological parameters of nonalcoholic steatohepatitis (NASH). After adjustment for age, gender, diabetes and alcohol consumption, the minor allele of rs738409C/G, a nonsynonymous coding SNP in the PNPLA3 (adiponutrin) gene encoding an I148M change, was associated with steatosis (p=0.03), portal inflammation (p=2.5x10-4), lobular inflammation (p=0.005), Mallory-Denk bodies (p=0.015), NAFLD activity score (NAS, p=0.004) and fibrosis (p=7.7x10-6). Two other SNPs in the SAMM50-PNPLA3 cluster demonstrated similar associations. Three SNPs on chromosome 10 in the CPN1-ERLIN1-CHUK region were independently associated with fibrosis (p=0.010). In children, no SNP was associated with histological severity. However, the rs738409 G allele was associated with younger age at the time of biopsy in multivariate analysis (p=0.045). In this large cohort of histologically-proven NAFLD, we confirm the association of the rs738409G allele with steatosis and describe its association with histological severity. In pediatric patients, the high-risk rs738409G allele is associated with an earlier presentation of disease. We also describe a hitherto unknown association between SNPs at a chromosome 10 locus and the severity of NASH fibrosis. In a subsequent project we genotyped adult Caucasians from the same NAFLD cohort for another group of SNPs that are associated with liver enzyme activity. We demonstrated associations of SNPs near or in the genes for hydroxysteroid (17&#946;) dehydrogenase 13 (HSD17B13), RAR-related orphan receptor &#945; (RORA) and protein phosphatase 1, regulatory subunit 3B (PPP1R3B) with histological features of NAFLD, after adjustment for age, gender and BMI. In-depth genotyping near RORA, a nuclear receptor involved in control of circadian rhythm and metabolic functions, showed that SNPs that are associated with NAFLD are located in the putative promoter region of 2 of the 4 splice variants (variants 2 and 3) as opposed to SNPs upstream of other variants, suggesting that alternation in the relative expression of the different isoforms affects fat accumulation in the liver. HSD17B13 is predominantly expressed in the liver and colocalizes with lipid droplets, but its substrate and physiological roles are unknown. In-depth genotyping of the gene region demonstrated associations of coding and splice-site SNPs in the gene with NAFLD, confirming a possible role for this enzyme in the pathogenesis of NASH. To better understand the roles of RORA and HSD17B13 in NAFLD and normal physiology, we studied their effect on fat accumulation in adipocytes and hepatocytes in culture. We found that siRNA knock-down of the expression of either RORA or HSD17B13, did not affect the degree of hepatic lipid accumulation under standard conditions. However, when cells were overloaded with nutrients (high glucose, high-fatty acids medium), the amount of intracellular fat significantly decreased with knock-down, predominantly through a decrease in the average size of the lipid droplets. A similar effect on adipocytes was seen by RORA, but not HSD17B13 knock-down. We have further undertaken studies in a mouse model of diet-induced NAFLD, where we have shown that knock-down of RORA expression in the liver diminishes hepatic fat accumulation, all supporting the role of RORA as an important regulator of excess hepatic fat storage. We are currently undertaking an effort to develop a liver-specific RORA knock-out strain to allow for more detailed characterization. Excess accumulation of fat in the liver is typically a feature of obesity and the metabolic syndrome and reflects caloric overload, and the fat accumulation could render the liver more susceptible to injury from oxidative stress and medications. On the other hand, fat accumulation itself is one of the myriad manifestations of drug-induced liver injury. To understand the interaction between liver fat and drug toxicity, we are evaluating the results of imaging studies, blood tests and clinical outcomes in 298 NIH patients who underwent chemotherapy to treat lymphoma. In this ongoing project we are examining whether chemotherapy induced fat accumulation on the one hand, and whether pre-existing fatty liver affected treatment outcomes and increased the risk for drug toxicity. Future studies in NASH will be directed at other means of improving this liver disease and understanding its pathophysiology. We are developing metabolic and physiologic studies of patients with NAFLD and/or metabolic syndrome and continue to study the functional significance of genetic variants and their role in disease pathophysiology and response to treatment. We are also continuing to study the prevalence and impact of fatty liver in unique patient populations.